Three Cheers for GCaMP : Optogenetic Brain Reading

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Three papers are out online in Nature Methods that show big improvements in calcium imaging with genetically encoded sensors. They are are based on the fluorescence intensity indicator, GCaMP. GCaMP, first developed by Junichi Nakai, consists of a GFP that has been circularly permuted so that the N and C termini are fused and new termini are made in the middle of the protein. Fused to one terminus is calmodulin and the other is a peptide, M13, that calmodulin (CaM) binds to in the presence of calcium. The name is supposed to look like GFP with a CaM inserted into it, G-CaM-P. Normally the GFP is dim, as there is a hole from the outside of its barrel into the chromophore. Upon binding calcium, this hole is plugged and fluorescence increases.

The first paper, A genetically encoded reporter of synaptic activity in vivo, from Leon Lagnado’s group, targets GCaMP2 to the outer surface of synaptic vesicles. This localization allows the fluorescence signal to be confined to the presynaptic terminal, where calcium fluxes in response to action potentials are high. This targeting improves the response magnitude of GCaMP2 and permits the optical recording of synaptic inputs into whatever region of the brain one looks at. They demonstrate the technique in live zebrafish.

In the second paper, Optical interrogation of neural circuits in Caenorhabditis elegans, from Sharad Ramanathan’s group, GCaMP2 has been combined with Channelrhodopsin-2 to perform functional circuit mapping in the worm. Since the worm’s structural wiring diagram has been essentially solved, functional data could say much about how “thick” the wires between each cell are. Unfortunately, with GCaMP2, the responses are too slow and weak to distinguish direct from indirect connections.

Finally, we have published a paper, Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators, describing the improved GCaMP3. This indicator has between 2-10x better signal to noise than GCaMP2, D3cpv and TN-XXL, depending on the system you are using. It’s kinetics are faster and it is more photostable than FRET indicators, and the responses are huge. When expressed in motor cortex of the mouse, neuronal activity is easily seen directly in the raw data. Furthermore, the sensor can be expressed stably for months, making it a potential tool for observing how learning reshapes the patterns of activity in the cortex.

Imaging of mouse motor cortex (M1) expressing the genetically-encoded calcium indicator GCaMP3 through a cortical window. After 72 days of GCaMP3 expression, large fluorescence transients can be seen in many neurons that are highly correlated with mouse running.

GCaMP3 is not perfect. It cannot reliably detect single action potential in vivo in mammals, though I doubt that any existing GECI can. Work continues on future generations of GCaMP that may achieve 100% fidelity in optical reading of the bits in the brain. However, there is considerable evidence from a number of groups that have been beta-testing the sensor, including the Tank lab of “quake mouse” fame, that it is a significant leap forward and unlocks much of the fantastic and fantasized potential of genetically-encoded calcium indicators.

Comparison of fluorescence changes in response to trains of action potentials in acute cortical slices.

I will try to post a more complete writeup of GCaMP3 for Brain Windows soon, with an unbiased eye to its strengths and weaknesses. We worked very hard to carefully characterize this sensor’s effects on cellular and circuit properties. If you have any questions about GCaMP3, please post them to the comments.

26 responses

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miko(07:25:01) :

I thought your blog had gone dead… I am loving GCaMP3 with a minor problem… the dynamic range is so huge that “quiet” cells often go below background and get subtracted to 0 in analysis, this creates some analysis headaches. But that’s really complaining about too much of a good thing.

Andrew, we have ben dreaming of this since 1994!
Congratulations.
will there be a “H-line” like GECI mouse soon?
will this approach allow enough protein for enough signal?
can you measure [Ca] yet?
if not, is this a goal, is imaging activity the main idea?
With the increase in affinity does this not going the wrong way? Lower affinity makes a better Ca dye (eg. fura-2 and fura-2FF).
in previous papers Svoboda measured [Ca] with dye in the same neuron as the GECI, how does GCaMP3 stack up in such conditions?

Graham,
1) What is a H-Line?
2) We haven’t made GCaMP3 transgenics yet, but other people are. I don’t know till we see how they turn out. Virus injection into cre lines works well for spatial and cell type restriction and shows great responses.
3) I think that quantitative calibration of these probes in vivo still requires assumptions that make me less than fully confident in believing absolute calcium level numbers. Each cell has different max dF/F from action potential trains, and there are a variety of reasons why that may be the case.
4) Using Ca as a reasonable and sensitive proxy for action potentials is the focus.
5) Define “better’. To pick up single action potentials, we need higher affinity. Ideally from a faster on-rate. Higher affinity probably means longer decay times but if we can get consistent 1AP from it, that’s probably a reasonable sacrifice in brain regions with sparse coding. There are potential issues with Ca buffering at higher affinities. GCaMP4 development may fork into different classes (a high affinity 1AP version and a fast, lower affinity version).
6) We haven’t done tons of experiments with dye combined with GC3 but check out sup fig 11b. you can infer the relative fluorescence change, although SNR of dyes is usually better due to higher brightness. Fluo4 picks up more spikes in bulk loading conditions than GC3.

H-line: this the eYFP mouse that Gan uses for his in vivo work.
For a static image of H-line:http://ellisdavieslab.googlepages.com/invivoimaging
3D movie:http://www.prairie-technologies.com/ (lower right)
Svoboda uses M-line, both were made in 2000 by Sanes and are commercial from JAX.
The nice thing about H-line is that they are VERY bright, so one can reliably breed and image. My lab has not done in utero electroporation so I have no feeling for how reliable it is.
thanks-Graham

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Nik(13:59:13) :

is it totally specific to Ca 2+ or is there a affinity remaining for Mg 2+ ?

Nik,
There is leftover affinity for Mg 2+. Mg is a serious issue for proper calibration of the probe. See fig 2 panel F of our 2008 paper for how that effects the reported Kd values of GCaMP2. We didn’t do much work to kill the Mg sensitivity in GCaMP3. TN-XXL has been designed to be more resistant to Mg.

…I got the chance to play with it and it’s great! Just one thing: You did not by any chance derive the rough GC3 parameters (especially on and off rates and Hill coefficient) for the GC2 model you published in BCB?

Graham, I suspect the size of the intact GCaMP3 makes it a little too big to passively diffuse into the nucleus. To quote wikipedia, “Small particles (< 30 kDa) are able to pass through the nuclear pore complex by passive diffusion. Larger particles are also able to pass through the large diameter of the pore but at almost negligible rates." Adding an NLS does target GCaMP3 there effectively.

When we DO see GCaMP3 in the nucleus, without an NLS, it a sign that the cell's calcium handling is screwed. In those cases, it looks the N-terminus of the GCaMP has been cleaved off, see SI Fig 11c. This may allow passive import, reveal a silent NLS, and/or cause brightening of the FP.

i was reading the recent Tank paper using gcamp3 in hippocampus, and I was wondering about the intrinsic variance in the fluorescent signal? they tried to look at how much variance there was in firing rate of place cells over multiple traversals of the same place, but it seems to me that the variance in the fluorescent signal itself at the number of APs they were looking at (most likely greater than 10) would not allow them to properly address the question, as the signal that they are measuring is itself quite variable. Is this in fact the case, and how big of an issue do you think variance in fluorescent signal is for imaging events which have more than a few spikes?

Thanks, so maybe the problem is because the time of resolution is to wide and the GCamP should take and lose the the calcium in the middle of measure, I put the GCamP3 in the C terminal of a calcium channel and when I put the agosnist I don’t see response with a medium with or without calcium.

My system is HEK cells, The calcium solution has a concentration of 2 mM. The solution without calcium it suposed that only contain traces, when I add calcium the final concentration is 5 mM, the way in that add it, it is injecting with a hammilton syringe directly in the fluorometer with agitation. I’m buffering the calcium with EGTA.
I have validated my system with FURA-RED and something that happend with my construction is that I see response with lysed the cells with triton 10%, thing that make me think that at least the GCamP is functional.
In the video of this blog you can see that the intensity of CCamP is transient, because of that make me think that when I see the GCamP together with the calcium channel in the membrane it should be a shorter peak of fluoresence.

I am trying to use GCamp3 in acute hippocampals slices, after delivery in vivo delvery/ expresion via AAV vector.

The purpose is to be able to conduct fast-confocal calcium imaging in acute slices of adult rodent, as chemical calcium indicators do not load well past the 3rd postnatal week. I can obtain strong GCamp3 expression, but the expression is not restricted to the cytoplasm of the most cells and we have difficulty finding cells that respond to stimulation (whether chemical or electrical). The few cells that do respond have the characteristic cytoplasmic localization.

My lab has extensive experiences w/ slice physiology, and our slices are typically very viable. Could something about acute slice preparation render GCamp3 nonfunctional? Or could high GCamp3 expression lessen the viability of acute slices from adult rodents?

Hi,
I am testing the effect of different drugs by in vivo calcium imaging in drosophila brains using the GCaMP3 sensor. Some drugs change the pH of the incubation medium. I have read that GCaMP is pH sensitive. Do you have any data on pH vs fluorescence in vivo? How fast are such changes? Will small changes in pH within the physiological range be a problem (pH 7.2 vs 7.4?). How will a change from pH7.4 to pH 4.0 affect the fluorescence?
Thanks!

A lower intracellular pH will protonate the chromophore, quenching fluorescence. An extracellular pH shift should have no effect unless that then affects the intracellular pH. Off the top of my head, I would expect a shift in intracellular pH from 7.4 to 7.2 would cause a drop of less than 10% fluorescence, while a shift from 7.4 to 4.0 would quench fluorescence by over 95%. Fluorescence shifts will track pH on the millisecond timescale. I don’t have data on how various drugs applied extracellularly will affect intracellular pH in a drosophila prep.